Bacterial viability and metabolic profiles of Lacticaseibacillus casei AP and Pediococcus acidilactici BE under various thermal treatment conditions

In this study, the effects of heat stress on the viability and metabolic profiles of two indigenous probiotic strains, Lacticaseibacillus casei AP and Pediococcus acidilactici BE, were assessed using liquid chromatography–high-resolution mass spectrometry. Both strains were subjected to thermal treatment at 37, 55, and 67 °C, followed by viability assessment and untargeted metabolomics analysis. The results indicated that compared to P. acidilactici BE, Lacticaseibacillus casei AP exhibited superior tolerance and maintained greater viability under heat stress. At 55 °C and 67 °C, Lacticaseibacillus casei AP resulted in lower reductions in cell viability, which was supported by adaptive responses involving heat shock proteins and membrane lipid modification. Heatmap visualization, principal component analysis, and partial least squares discriminant analysis revealed distinct metabolite signatures across all strains and temperature conditions. In P. acidilactici BE, metabolites such as citric acid, tri (2-ethylhexyl) ester, N-(3-aminopropyl) hexadecanamide, and valine were prominent under stress, whereas Lacticaseibacillus casei AP exhibited increased production of nucleotides (e.g., guanosine-5′-monophosphate), peptides (e.g., rhabdopeptide-1), and membrane stabilizing compounds (e.g., monogalactosyl diacylglycerols and sterols). These findings confirm the occurrence of strain-specific metabolic adaptations, with Lacticaseibacillus casei AP demonstrating a clear protective mechanism against heat stress. This study provides critical insights into probiotic resilience and offers guidance for the development of stable probiotic formulations capable of withstanding industrial processing and storage at elevated temperatures.